Pharmaceutical preparation comprising vWF propeptide

ABSTRACT

Described is a pharmaceutical preparation for treating blood coagulation disorders comprising an effective amount of vWf propeptide as well as a method for producing such a prepartion.

This application is the U.S. national phase of PCT/EP98/03090 filed May26, 1998, which claims priority from the Austrian application A 917/97,filed May 28, 1997.

FIELD OF THE INVENTION

The invention relates to a pharmaceutical preparation comprising thevWF-propeptide (pp-vWF).

BACKGROUND OF THE INVENTION

Von Willebrand factor (vWF) is a glycoprotein circulating in plasma as aseries of multimers ranging in size from about 500 to 20,000 kD.Multimeric forms of vWF are composed of 250 kD polypeptide subunitslinked together by disulfide bonds. vWF mediates the initial plateletadhesion to the sub-endothelium of the damaged vessel wall, only thelarger multimers also exhibiting hemostatic activity. It is assumed thatendothelial cells secret large polymeric forms of vWF and that thoseforms of vWF which have a low molecular weight (low molecular weightvWF) have arisen from proteolytic cleavage. The multimers having largemolecular masses are stored in the Weibel-Pallade bodies of theendothelial cells and liberated upon stimulation.

vWF is synthesized by endothelial cells and megakaryocytes as prepro-vWFthat consists to a large extent of repeated domains. Upon cleavage ofthe signal peptide pro-vWF dimerizes through disulfide linkages at itsC-terminal region. The dimers serve as protomers for multimerizationwhich is governed by disulfide linkages between the free end termini.The assembly to multimers is followed by the protelytic removal of thepropeptide (Leyte et al., Biochem. J. 274 (1991), 257–261).

The full length of cDNA of vWF was cloned; the propolypeptidecorresponds to amino acid residues 23 to 764 of the full lengthprepro-vWF (Eikenboom et al (1995) Haemophilia 1, 77–90).

The propeptide of vWF (pp-vWF) was shown to be identical to the vonWillebrand antigen II, the second identified antigen that is deficientin the plasma and platelets of patients with severe von Willebranddisease (vWD). pp-vWF is specifically localized in platelets sinceplasma contains less than 5% of total propeptide vWF in blood, assumingthe platelet count is 3×10⁸ per ml. As already known, pp-vWF is releasedfrom platelets upon activation by various agonists. The pp-vWF is aglycoprotein not only because it reacts with periodic acid Schiff'sreagent but also because it binds to lentil lectin. pp-vWF bindsspecifically to native type I collagen, but does not bind toheat-denatured collagen. It was shown that the affinity between pp-vWFand type I collagen was quite high so that the binding—which does notrequire any divalent cation and is not affected by addition of a peptidethat contains sequence of arginine-glycine-aspartic acid (that is knownto inhibit many cell attachment processes)—rapidly reached equilibrium.

The physiological role of pp-vWF is postulated to lie in the governmentof the assembly of vWF multimers, either before or after the cleavagefrom pro-vWF molecules. (Takagi et al., JBC 264 (18) (1989),10425–10430).

pp-vWF was also shown to inhibit the platelet collagen interactionaction (Takagi et al., JBC 264(11) (1989), 6017–6020).

In Isobe et al. (JBC 272 (13) (1997), 6447–6453) the role of pp-vWF as anovel physiological ligand and an adhesion substrate for α4β1integrin-expressing leukemia cells was investigated. It was found thatpp-vWF plays an important role in the mechanism underlying the melanomametastasis as well as vascular inflammation.

Although pharmaceutical preparations containing mature vWF are known(see e.g. U.S. Pat. No. 5,571,784) the pharmaceutical usage of pp-vWF orthe pro-form vWF have not been described or suggested in the prior art.According to the U.S. Pat. No. 5,571,784 vWF does not impair thesystemic anticoagulatory effect of the anticoagulant hirudin as measuredby the aPTT, rather it decreases the bleeding side effects ofanticoagulant therapy. vWF is therefore proposed as a pseudo-antidote inassocation with hemorrhages which are produced by administeringantithrombotic and/or fibrinolytic agents.

From Blann et al. (Eur. J. Vasc. Surg. 8 (1994), 10–15) it is also knownthat vWF levels are increased with risk factors for atherosclerosis andin patients with diffuse arterial disesase. The level of vWF is alsothought to be a measure of endothelial ₄>integrin-expressing leukemiacells was investigated. It was found that pp-vWF plays an important rolein the mechanism underlying the melanoma metastasis as well as vascularinflammation.

SUMMARY OF THE INVENTION

It is the object of the present invention to provide a vWF pharmaceuticwith improved properties. The preparation should enhance the intrinsicblood coagulation activity in individuals and reduce the arterialthrombotic risk of vWF therapy.

The object is solved by the present invention by providing apharmaceutical preparation for treating blood coagulation disorderscomprising an effective amount of vWF propeptide. It was found out thatpp-vWF plays an essential role in blood coagulation. It promotes theintrinsic blood coagulation and thereby acts on secondary hemostasis. Atthe same time it inhibits the platelet adhesion and controls the primaryhemostatic activity of mature vWF by binding to collagen. Based on thesefindings, a vWF preparation can be improved providing additional pro-vWFor pp-vWF as a separate effective protein in the vWF preparation. pp-vWFcontrols the primary hemostatic activity of the mature vWF and thusreduces the potential thrombotic risk of vWF, for example inducingarterial thrombosis as indicated by the prior art.

It was surprisingly found out that a recombinant vWF preparation (rvWF)containing substantial amounts of pp-vWF induces an elevated thrombingeneration in vitro. The thrombin generation was measured by an in vitroassay performed with washed vWD platelets in FVIIIC substituted vWDplasma upon addition of FEIBA (factor VIII inhibitor bypassing activity;Austrian patents No 350 726, 368 883 and 398 079) to initiate theactivation of prothrombin. When the rvWF preparation contained apredominant amount of pro-vWF and pp-vWF the thrombin generation wassubstantially increased. The contribution of the pp-vWF to thecoagulation-promoting effect of vWF is therefore surprisinglysignificant.

Since these results imply that the effect of vWF as blood coagulationfactor is related to pp-vWF, the pharmaceutical preparation based on thepp-vWF or pro-vWF is applicable in areas wherein vWF has been proposedand even more areas where a coagulation-promoting effect is desired.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of pro-vWF on the thrombin generation in plasmain the presence of platelets.

FIG. 2 shows the dose dependence effect of pro-vWF on the thrombingeneration in plasma in the presence of platelets.

FIGS. 3 a and 3 b show the comparison of the in vivo effect of pro-vWFand plasma derived vWF in a dog.

DETAILED DESCRIPTION

The preparation of the pp-vWF or pro-vWF is well known in the art due tomany papers relating to expression and properties of vWF or fordiagnostic purposes, see e.g. experimental procedures in Isobe et al.,Takagi et al. (both 1989 papers) or Leyte et al. However, there was notyet any individual coagulation promoting activity reported to beassociated with vWF and the propeptide region of vWF and therefore not arespective pharmaceutical preparation. Since the pp-vWF is a welldefined polypeptide and easily prepared by recombinant DNA-technology,the recombinant way of production is a preferred embodiment forproducing the present pharmaceutical preparation. The polypeptide mayalso be purified or purified further by chromatography, such as by ionexchange chromatography and/or affinity chromatography using monoclonalantibodies, heparin, collagen, factor VIII protein, or fragments thereofas affinity ligands. It is also possible to separate pp-vWF fromcontaminating proteins and/or mature vWF by gel filtration.

A preferred embodiment of the present invention is a pharmaceuticalpreparation essentially consisting of the vWF-propeptide. Thereby thepreparation contains purified pp-vWF or pro-vWF to the extent that atleast 8.0%, preferably at least 90%, most preferred more than 95% orabout 100% pure pp-vWF or pro-vWF is contained.

Another embodiment of the present invention is a preparation essentiallyconsisting of the pro-vWF containing the vWF-propeptide as apharmaceutical preparation.

As stated above it is preferred to use recombinantly produced pro-vWF orpp-vWF for the present preparation. (FEBS Letters 351 (1994), 345–348 orBlood 88 (8) 19996, 2951–2958)

The preparations of the present invention preferably contain at least 10nM pp-vWF, more preferably at least 30 nM, especially more than 50 nM,and/or at least 10 nM pro-vWF, more preferably more than 100 nM,especially more than 250 nM. The effective amount is defined to obtain app-vWF level of at least twice the physiologic amount in human plasma.In Blood 88 (8) 1996, 2951–2958 it is reported that the molar ratio ofthe propeptide and vWF concentration is about 0,1 in Normal Plasma. Thepp-vWF is considered as a rather abundant protein at a concentration of5 to 100 nmol/L depending on the state of activation of the endothelium.

If vWF is contained in the preparation besides pro-vWF the molar ratioof the pro-vWF and vWF is at least 10%, which may be measured asU-Antigen detected by a polyclonal antibody preparation directed againstvWF antigen. In the preferred preparation according to the invention themolar ratio is even higher, at least 20% or more preferred at least 50%.The most effective preparation according to the invention contains morethan 80% of vWF-Antigen as pro-vWF.

It turned out that a pharmaceutical preparation according to theinvention based on pro-vWF is rapidly processed upon administration invivo. The thus generated pp-vWF is effective in its thrombin potentialand coagulation-promoting activity.

In another preferred embodiment the pharmaceutical preparation accordingto the present invention further contains a hemostasis protein,preferably a blood factor. Preferred embodiments of these blood factorsare selected from the group consisting of mature vWF, factor VIII,activated blood coagulation factors, blood factors with FEIB-activityand FEIBA. Any hemostasis protein decreasing the aPTT or PT of normalplasma is a suitable combination with the pp-vWF or pro-vWF.

A combination of the pp-vWF preparation with FVIIIC provides for apharmaceutical preparation with improved coagulation activity. When thepropeptide is in the form of pro-vWF that is complexed to FVIIIC, thepharmaceutical preparation according to the present invention showsadditionally improved FVIIIC-stability.

The further combinations in a pharmaceutical preparation according tothe invention are provided with a platelet component. Some of thecomponents having binding properties or activity to vWF or pro-vWF orpp-vWF, which are suitable to contribute to the physiologicalactivities, are collagen, platelet glycoprotein, a platelet, fibrinogen,fibrin, heparin, or a derivative thereof.

The pharmaceutical preparation of the present invention may also furthercontain phospholipids.

The pharmaceutical preparation according to the present inventionpreferably has been made virus safe by treating for virus inactivationor removal.

The virus inactivation or removal treatment may be performed by anytreatment accepted as being efficient. According to preferredembodiments of the present invention the pharmaceutical composition istreated with tensides and/or heat, e.g. by a heat treatment in the solidstate, especially a steam treatment according to EP 0 159 311 or EP 0519 901 or EP 0 637 451, by a hydrolase treatment according to EP 0 247998, by a radiation treatment or by a treatment with chemical orchemical/physical methods, e.g. with chaotropic agents, according to WO94/13329, by a treatment with organic solvents and/or tensides accordingto EP 0 131 740 or photoinactivation. Nanofiltration also represents apreferred method of depleting viruses within the scope of the presentinvention.

The pharmaceutical preparation according to the present inventionfurther contains a pharmaceutically acceptable carrier and/or suitablebuffer auxiliary preserving and/or stabilizing substances likecarbohydrates or salts, or protease inhibitors or cofactors,respectively. The preparation is finally formulated for especiallyparenteral or topical uses like any known vWF preparation according tothe prior art. This may be done by filling it into containers in a formsuitable for administration and preferably packing it so as to bestorage stable, optionally in the lyophilized or frozen state.

The preparation according to the present invention may be produced bothby purification from blood serum or plasma and by a respectiveexpression system. Transgenic animals might as well provide the sourceof pp-vWF or pro-vWF.

A preparation according to the present invention may also be provided byexpression of the pp-vWF or the pro-vWF containing pp-vWF in vivo or exvivo. Especially suitable for such procedure are cells derived frommammals, in particular human cells, which can be cultured or employed inhuman gene therapy. Also transformed cells expressing pp-vWF or thepro-vWF as a heterologous protein are a suitable source for obtainingthe preparation according to the invention.

A further embodiment according to the present invention is also apharmaceutical preparation containg a pro-vWF mutant with a mutation atthe cleavage site. Such a mutant has been described by Borchiellini etal. for experimental purposes (Blood 88 (8), 2951–2958 (1996)). Thedescribed vWF-Gly 763 has a mutation which provides for a pro-vWF thatis unclearable by physiological enzymes. The resistance of the pro-vWFagainst cleavage leads to the prolonged half-life of thecoagulation-promoting activity of the pp-vWF being comprised in thepro-vWF form. Thereby a prolonged action is designed by a specificmutation at the cleavage site.

This specific mutation might by effected by the techniques of Lankhof etal. (Thrombin and Haemostasis 77 (5), 1008–13 (1997)) who produced adeletion mutant lacking the A2 domain, which was resistant toproteolysis unless it became sensitive upon unfolding to the molecule.

Other mutant proteins of pp-vWF or pro-vWF that exhibit the propertiesof the native proteins may also be used for the present preparations. Inthis case it is preferred to employ an analogue or mutant having atleast 80% homology and the function to act as a modified pp-vWF orpro-vWF.

Yet another effect of the present invention is a method for producing apharmaceutical preparation containing an effective amount of pp-vWFcomprising providing a source material containing the vWF propeptide,separating the pp-vWF from the source material and formulating thepp-vWF to a pharmaceutical preparation.

Source material may preferably be blood, serum, blood fractions,colostrum or milk of transgenic animals, or cell culture solutions,especially from cells that have been produced by recombinantDNA-technology. The source material containg pp-vWF preferably containsthe pp-vWF in a pro-vWF. Methods and techniques are described in FEBSLetters 351, 345–348 (1994) or Borchiellini et al. supra.

The expression is preferably performed in a way to prevent theprocessing and maturation of vWF to obtain the pro-vWF. This may beeffected by the omission or inhibition of processing enzymes. Theinhibition of processing enzymes like furin or PACE or the multimeraseas described in A 770/96 and 769/96 prevents the premature processing ofpro-vWF to vWF. On the other hand the pp-vWF might be expressed as aseparate protein or obtained upon cleavage and processing of the pro-vWFin vitro.

Yet another preferred embodiment of the present invention is conductingthe method by providing a source material containing the pro-vWF as amutant pro-vWF with a mutation at the cleavage site of the pp-vWF, suchas an amino acid change at 763 like pro-vWF-Gly 763.

Alternatively, the pharmaceutical preparation may be produced in thepresence of an inhibitor inhibiting the cleavage of the pp-vWF from thepro-vWF. Examples for such inhibitors are antibodies against thecleavage site or a binding peptide directed against the cleavage site orinhibitor of processing enzyme.

As stated above, the preparation and separation steps of pp-vWF arewell-known in the art due to various reports of the experimentsconducted with pp-vWF (see Isobe et al., Tagaki et al., Leyte et al.).

Of course, the method according to the present invention preferablyexhibits a treatment for inactivating or removing viruses, since thepp-vWF is a biological protein and in a form which is administered tohumans.

The invention further provides pp-vWF and/or pro-vWF for use as amedicine. The effective dose to elevate the pp-vWF level in vivo to atleast twice the physiological amount may be provided by administeringthe pp-vWF or the pro-vWF once or several times a day. Due to the rathershort half-life of the pp-vWF in vivo it might be necessary toadminister the protein frequently during the acute disorder.

Yet another aspect of the present invention is the use of pp-vWF and/orpro-vWF containing the pp-vWF for the preparation of a pharmaceuticalcomposition for treating a patient at a risk of blood coagulationdisorders, such as vWD, hemophilia (e.g. phenotypic hemophilia,hemophilia A and factor VIII inhibitors).

The effective dosage of the preparation when applied will vary dependingon the respective syndrome and preferably should be chosen afterdetermination of the blood levels of the critical blood factors or riskfor thrombosis in the patient. The optimum dosage also depends onwhether or not the parenteral, preferably intravenous, subcutaneous orintramuscular administration is to be effected in bolus form or as adepot. By using a suitable carrier material such as liposomes a peroraladministration is feasible. It also depends on whether it is to beapplied systemically and/or locally at the site of the blood coagulationdisorder.

Therefore, the invention also provides for a method of treating apatient at a risk of blood coagulation disorders comprisingadministering to said patient an effective amount of vWF-propeptide orpro-vWF. Preferably, a patient suffering from vWD, phenotypichemophilia, hemophilia A or factor VIII inhibitors is treated accordingto the invention.

Due to the positive properties pp-vWF or pro-vWF exhibit when combinedwith preparations with a risk for arterial thrombosis, such asvWF-preparations, it is another aspect of the present invention to usepp-vWF or pro-vWF to reduce thrombosis risk in vWF-preparations. Therebythe potential exaggeration of arterial thrombus formation is effectivelydown-modulated, whereas the intrinsic and extrinsic blood coagulation ispromoted in case of a coagulation deficiency.

In particular the compatibility of a vWF preparation is ameliorated andimproved by the addition of and combination with the pp-vWF or pro-vWFin effective amounts. Because of the controlling function of the vWFpropeptide it further contributes to the treatment and prevention ofadverse reactions of endogenous and exogenous vWF, particularly elevatedvWF levels in patients associated with thrombotic thrombocytopenicpurpura, Henoch Schönlein purpura, preclampsia, neonatalthrombocytopenia or hemolytic uremic syndrome, myocardial infarction ora poor outcome following arterial surgery.

The present invention will be explained in more detail by way of thefollowing examples and drawing figures to which, however, it shall notbe restricted.

EXAMPLES

1. The Effect of provWF an ppvWF on the Thrombin Generation in Plasma inthe Presence of Platelets.

Severe vWD plasma (George King Bio-Medical Inc., USA) which waspreviously reconstituted with 1 U/ml FVIIIC (Recombinate, Baxter, USA)(200 μl) was incubated with 50 μl washed platelets from a severe vWDpatient (Type III) in the presence of 0.1 U/ml FEIBA (Immuno, Austria)and 16 mM CaCl₂. Alternatively FVIIa could also be used as an activator,preferably in a final concentration of 0.2 μg/ml. Subsamples of 10 μlwere withdrawn at time intervals, and added to 300 μl chromogenicsubstrate for thrombin (D-cyclohexyl-gly-L-Ala-L-Arg-pNA; Immuno,Austria) containing 3 mM EDTA to stop any further reactions. Thereaction was stopped by the addition of 100 μl of 75% (v/v) acetic acid,and the absorbance, which is the function of the thrombin concentration,was measured at 405 nm.

Two different vWF preparations (containing lower and higher amounts ofprovWF and propeptide) were added to the factor VIII reconstitutedplasma samples in a final concentration of 1 Risto U/ml, and thrombingeneration was followed. (1 Risto U/ml=the ristocetin cofactor activityof the vWF in normal plasma). Thus the plasma samples contained in thecase of the preparation with the low provWF and low propeptide 0.24 nMprovWF and 0.19 nM propeptide, while the other one contained 34 nMprovWF and 4 nM propeptide, respectively.

The results are depicted on FIG. 1.

It is clear from the curves, that albeit both preparations increased therate of thrombin generation, the high provWF and high propeptidecontaining product had a stronger effect and produced the enhancedthrombin generation.

2. The Effect of provWF on the Thrombin Generation in Plasma in thePresence of Platelets.

The effect of a recombinant vWF preparation, which contained more then90% provWF of the total vWF antigen on the thrombin generation wasinvestigated in the system described by the Example 1. Washed plateletsfrom an other severe vWD patient (Type III) and the isolated plasmasample of the same patient, reconstituted with 1 U/ml FVIIIC were usedin the experiments. Recombinant provWF of 2 and 5 vWF-Ag U/ml (1 AgU=the vWF antigen amount in 1 ml normal plasma) respectively, were addedto the plasma samples and thrombin generation was compared with that ofin the absence of vWF. FIG. 2 shows, that above a threshold of 2 vWF-AgU/ml an increased thrombin generation was observed.

Example 3 Comparison of the In Vivo Effect of a High provWF ContainingRecombinant and a Plasma Derived vWF Preparation

A vWF deficient dog (vWF antigen below the detection limit, and factorVIII activity about 50% of the normal) was anesthetized and infused with35 Risto U/ml of the recombinant vWF used in the example 2, containingmore then 90% of provWF. Prior to the infusion and 15, 30, 40 minutes,1, 2, 3, 6, 24, 48, 72, an 95 hours post infusion plasma samples weretaken. 95 hours later a plasma derived vWF preparation (HaemateHS—Behringwerke, Marburg, Germany) was added, and plasma samples weretaken again at the same intervals after the infusion, as before. Thisplasma derived vWF preparation further contained Factor VIII:C, but noprovWF and no ppvWF. The plasma samples were analyzed for total vWFantigen (Asserachrom ELISA, Boehringer), provWF and propeptide antigen(according to Borchiellini at al Blood 88, 2951, 1996), as well as forthe thrombin generation potential, as described in Example 1. Thethrombin potential is defined as the maximum thrombin concentrationmeasured as described in Example 1.

FIG. 3 shows the correlations of the various parameters, expressed aspercent of maximum, regarding the initial values as zero.

Thrombin potential increased in parallel with the increase of propeptideafter the treatment with a recombinant pro-vWF preparation. ELISAresults showed, that a few percent of pro-vWF remained in thecirculation after 15 minutes, and it could no longer be detected (datanot shown), but a significant increase in the propeptide and vWF wasobserved. In contrast, no propeptide and also no substantial thrombinpotential was observed in the dog after the plasma derived vWF infusion,despite the vWF antigen level increase.

1. A pharmaceutical preparation for treating blood coagulationdisorders, said preparation comprising at least 10 nM von WillebrandFactor (vWF) propeptide and a hemostasis protein, wherein saidpreparation has been treated for at least one of virus inactivation andvirus removal so the preparation is suitable for therapeuticadministration.
 2. A preparation as set forth in claim 1, wherein saidhemostasis protein is a blood factor.
 3. A preparation as set forth inclaim 2, wherein said blood factor is selected from the group consistingof mature vWF, factor VIII, activated blood coagulation factors, andblood factors with factor VIII inhibitor bypassing activity.
 4. Apharmaceutical preparation for treating blood coagulation disorders,said preparation comprising at least 10 nM von Willebrand Factor (vWF)propeptide and a phospholipid, wherein the preparation has been treatedfor at least one of virus inactivation and virus removal so thepreparation is suitable for therapeutic administration.
 5. Apharmaceutical preparation for treating blood coagulation disorders,said preparation comprising at least 10 nM von Willebrand Factor (vWF)propeptide and at least two components, wherein the components areselected from the group consisting of a blood factor, a plateletcomponent, and a phospholipid, wherein said preparation has been treatedfor at least one of virus inactivation and virus removal so thepreparation is suitable for therapeutic administration.